Testis-specific differentiation-regulatory factor

ABSTRACT

A gene expressed specifically in the testis has been unexpectedly isolated in the course of studies of the expression of a gene encoding an unknown protein that triggers cell death. The isolated gene was a novel gene sequence that had no significant homologue in the database. This gene was also found to be involved in the regulation of differentiation in the testis.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/876,841, filed Jun. 28, 2004, which is in turn a divisional ofinternational application number PCT/JP99/03859, with an internationalfiling date of Jul. 16, 1999, which entered the national stage in theU.S. under 35 U.S.C. § 371 as U.S. patent application Ser. No.09/743,237 on Jun. 4, 2001, now U.S. Pat. No. 6,835,813 and furtherclaims the benefit of Japanese Patent Application No. 10-219856, filedJul. 17, 1998.

All priority documents, including the specification, drawings, claimsand abstract, are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a protein and its gene involved in thedifferentiation of testicular cells and belongs to the field ofbioscience, specifically, developmental biology.

BACKGROUND OF THE INVENTION

In the developmental process, reproductive cells carry outspermatogenesis via a differentiation process that includes meiosis.This differentiation process is different from that of somatic cells andconsists of three main steps. The first step is the proliferation ofspermatogenous cells and differentiation into primary spermatocytes. Thesecond is the meiosis of primary spermatocytes, and the third is thetransformation into sperms.

Owing to the progress in Molecular Biology, recent years have seen theisolation of several genes specifically expressed in these stages. Forexample, Hox-1.4 (Propst, F. et al. (1988) Oncogene 2:227-33), ferT(Sarge, K. D. et al. (1994) Biol Reprod 50:1334-1343) of the HSP70family, and TESK1 (Toshima, J. et al. (1995) J. Biol. Chem.270:31331-31337) that is a serine-threonine kinase, have been reportedas genes specific to primary spermatocytes. However, still very littleis known about the biological and physical roles of their gene products.

Genes expressing specifically in the differentiation process ofreproductive cells carry a fundamental and vital role that decides thefate of those cells, and thus, defects in these genes are considered tobe a cause of diseases such as infertility. Therefore, genes expressingspecifically in the differentiation process of reproductive cells arerecently gaining wide attention as targets in the development ofpharmaceutical drugs. Such drugs can be used for the prevention andtreatment of diseases such as infertility caused by defects inreproductive cell differentiation.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a novel gene and proteininvolved in differentiation of testicular cells. It also provides avector and a transformant useful in producing the protein as well as amethod of producing the protein. The present invention further providesan oligonucleotide useful in the isolation and the detection of the geneof the invention.

In one embodiment, the invention provides an isolated DNA encoding aprotein comprising SEQ ID NO: 4 or 5.

In another embodiment, the invention provides a vector comprising a DNAencoding a protein comprising SEQ ID NO: 4 or 5.

In a further embodiment, the invention provides a transformantcomprising a DNA encoding a protein comprising SEQ ID NO: 4 or 5.

In yet another embodiment, the invention provides a method of producinga protein comprising culturing a transformant comprising the DNAencoding a protein comprising SEQ ID NO: 4 or 5 under conditions wherethe transformant encodes a protein and obtaining the protein from thetransformant or the culture supernatant thereof.

In a further embodiment, the invention provides an isolated DNAspecifically hybridizing to a DNA comprising the nucleotide sequence ofany one of SEQ ID NOs: 1 to 3, and comprising at least 15 nucleotides.

In still another embodiment, the invention provides an isolated DNAencoding a protein which comprising an amino acid sequence in which oneof several amino acids of SEQ ID NO: 4 or 5 have been replaced, deleted,and/or added, and which is functionally equivalent to a proteincomprising SEQ ID NO: 4 or 5.

In yet a further embodiment, the invention provides an isolated DNAencoding a protein which is encoded by a DNA hybridizing under stringentconditions to the DNA comprising the nucleotide sequence of SEQ ID NO: 1or 3, wherein the protein has testicular differentiation activity.

In a further embodiment, the invention provides a method of producing aprotein comprising culturing a transformant comprising a DNA underconditions wherein a protein is expressed, wherein the protein comprisesan amino acid sequence in which one of several amino acids of SEQ ID NO:4 or 5 have been replaced, deleted, and/or added, and wherein theprotein is functionally equivalent to a protein comprising SEQ ID NOs: 4or 5; and obtaining the protein from the transformant or the culturesupernatant thereof.

In yet another embodiment, the invention provides a method of producinga protein comprising culturing a transformant comprising DNA whichhybridizes under stringent conditions to a DNA comprising a nucleotideof SEQ ID NO: 1 or 3 under conditions where a protein is expressed andobtaining the protein from the transformant or the culture supernatantthereof.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory only,and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an electrophoretic photograph showing the results of Northernblot analysis of Tesmin gene expression in various mouse tissues.

FIG. 2 is an electrophoretic photograph showing the results of Northernblot analysis of Tesmin gene expression in various human tissues.

FIG. 3 is an electrophoretic photograph showing the results of themolecular weight detection of mouse Tesmin protein expressed by in vitrotranslation.

FIG. 4 is an electrophoretic photograph showing the results of Northernblot analysis of Tesmin gene expression in the testis of ICR strainmouse at day 4, 8, 12, 18, and 42 following birth, and in the day 56testis of W/Wv strain mouse. “MEG1” and “ssH2B” were used as testisdifferentiation markers. “GAPDH” was used as the control.

FIG. 5 is a photomicrograph showing the results of detection of Tesmingene expression in testicular tissues by in situ hybridization.

FIG. 6 is a photomicrograph and schematic diagram of the chromosomallocation showing the results of the detection of Tesmin gene location inthe mouse chromosome using a probe specific to the Tesmin gene.

FIG. 7 is a photomicrograph and schematic diagram of the chromosomallocation showing the results of the detection of Tesmin gene location inthe human chromosome using a probe specific to the Tesmin gene.

FIG. 8 is a photomicrograph showing the intracellular localization ofthe complete Tesmin protein and its deletion mutant.

FIG. 9 shows the results of the detection of the Tesmin protein (fusionprotein with GST) by Western blotting using the prepared anti Tesminantibody. Detection using anti GST antibody was also done concurrently.“IPTG+” means the protein detected by addingisopropyl-β-D-thiogalactoside (IPTG) to cDNA-introduced E. coli toinduce the expression of the recombinant protein, subjecting the celllysate to SDS-PAGE and Western-blotting, and “IPTG−” means the proteindetected in a lysate of E. coli to which IPTG was not added.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel protein relating to thedifferentiation of testicular cells, and the encoding gene. It alsoprovides a vector and transformant used for, for example, producing theprotein, and a method of producing the protein. The present inventionalso provides an oligonucleotide used for the isolation and thedetection of the gene of the invention.

The inventors were evaluating the expression of genes encoding unknownproteins that trigger cell death when, irrelevant to their original aim,they unexpectedly succeeded in isolating a novel gene specificallyexpressed in the testis. When the databases were searched for theisolated gene, it was found to be a novel gene that did not have asignificant homologous gene. Structural analysis of the protein encodedby the gene showed that it had in part a structure similar to themetal-binding site of metallothionein, which is known to be ametal-binding factor. Expression analysis in tissues revealed that thegene is extremely specific to the testis, especially to primaryspermatocytes. The expression was not seen in the testis of infertilemice. Analysis of the human and mouse chromosomal locations showed thatthe gene was located in the same site as the gene locus that is known tobe defective in infertile mice. Results of these analyses suggest thatthe protein encoded by the isolated gene is involved in regulating thedifferentiation of the testis.

The present invention relates to a novel protein involved in theregulation of testicular differentiation having a metal-binding site,and the gene thereof, more specifically:

-   (1) a protein comprising the amino acid sequence of SEQ ID NO: 4 or    5;-   (2) a protein which comprises an amino acid sequence in which one or    more amino acids in the amino acid sequence of SEQ ID NO: 4 or 5    have been replaced, deleted, and/or added, and which is functionally    equivalent to the protein of (1);-   (3) a protein which is encoded by a DNA hybridizing to the DNA    comprising the nucleotide sequence of SEQ ID NO: 1 or 3, and which    is functionally equivalent to the protein of (1);-   (4) a DNA encoding the protein of any one of (1) to (3);-   (5) a vector comprising the DNA of (4);-   (6) a transformant comprising the DNA of (4) in an expressible    manner;-   (7) a method of producing the protein of any one of (1) to (3)    comprising the steps of culturing the transformant of (6), and    collecting the expressed protein from said transformant or the    culture supernatant thereof;-   (8) an antibody binding to the protein of any one of (1) to (3);    and,-   (9) a DNA specifically hybridizing to a DNA comprising the    nucleotide sequence of any one of SEQ ID NOs: 1 to 3, and comprising    at least 15 nucleotides.

The present invention provides the protein Tesmin, which may regulatethe differentiation of spermatogenous cells into primary spermatocytes,and the gene thereof.

The inventors isolated two types of Tesmin cDNA of mouse origin arisingpossibly from splicing differences in the transcriptional process. Thenucleotide sequences of these cDNAs are shown in SEQ ID NOs: 1 and 2,and the amino acid sequence of the protein encoded by these cDNAs in SEQID NO: 4. The nucleotide sequence of human Tesmin cDNA also isolated bythe inventors is shown in SEQ ID NO: 3, and the amino acid sequence ofthe protein encoded by the cDNA is shown in SEQ ID NO: 5.

As shown in SEQ ID NOs: 1 and 2, mouse-derived Tesmin cDNA has an ORFencoding a protein comprising 295 amino acids. On the other hand,human-derived Tesmin cDNA has an ORF encoding a protein comprising 299amino acids, as shown in SEQ ID NO: 3. SDS-PAGE analysis of the in vitrotranslational product of mouse Tesmin using ³⁵S-labeled methionineshowed that mouse Tesmin protein had a molecular weight of 32.5 kDa(FIG. 3).

Among the tissues within the body, both mouse and human Tesmin geneswere expressed only in the testis, as revealed by Northern blot analysisand RT-PCR (FIGS. 1 and 2). RT-PCR analysis showed that Tesmin gene ishardly expressed in the immature testis up to day 8 following birth, butthe expression increases from day 12 when the sperm differentiationstarts, and its high expression stabilizes from day 18. In the W/Wvmouse known as an infertile mouse that lacks the growth factor receptor“c-kit” gene, Tesmin gene expression was hardly seen even in the maturedtestis of day 52 following birth (FIG. 4). These facts suggest that theTesmin protein is involved in the differentiation of the testis. TheTesmin protein and its gene can be applied, for example, in thetreatment of infertility.

The Tesmin protein of the invention can be prepared by incorporating DNAencoding the protein (e.g., DNA comprising the nucleotide sequence ofany one of SEQ ID NO: 1 to 3) into a suitable vector, introducing thisinto a suitable host cell, and purifying the protein from thetransformant obtained. The protein of the present invention can also beprepared as a recombinant protein made using genetic engineeringtechniques by culturing cells transformed with DNA encoding the Tesminprotein, as mentioned later. The natural protein can be isolated fromtesticular tissues by methods well known to one skilled in the art, forexample, the affinity chromatography later described, using an antibodythat binds to the Tesmin protein.

A skilled artisan can prepare not only a natural Tesmin protein, butalso a modified protein functionally equivalent to the natural proteinby, for example, suitably performing amino acid substitution of theprotein using known methods. Amino acid mutations of a protein can occurspontaneously, too. Therefore, the protein of the invention includes amutant in which the amino acid sequence of the natural protein wasmutated by, for example, replacing, deleting, or adding one or severalamino acids, and which is functionally equivalent to the naturalprotein. Methods well known to a skilled artisan for modifying aminoacids are, for, PCR-mediated site-specific-mutation-induction system(GIBCO BRL, Gaithersburg, Md.), oligonucleotide-mediatedsite-specific-mutagenesis (Kramer, W. and Fritz, H J (1987) Methods inEnzymol. 154:350-367), the Kunkel method (Methods Enzymol. 85:2763-2766(1988)), and so on. The number of amino acids mutated is normally withinten amino acids, preferably within six amino acids, and more preferablywithin three amino acids.

Herein, “functionally equivalent” means that the mutant protein has abiochemical and/or biological activity equivalent to the naturalprotein. As such activities, for example, the binding activity betweenthe protein and metal, and the testicular cell differentiation-inducingactivity can be given.

The metal-binding activity can be detected, for example, as follows.First, the recombinant Tesmin protein is EDTA-treated to remove heavymetals that may be bound to the Tesmin protein. Next, EDTA is removed bygel filtration, and then, the heavy metals (for example, Zn²⁺, Cd²⁺,Cu²⁺, etc.) to be examined are added and reacted with the recombinantTesmin protein. After reacting, the presence or absence of a metal bondis detected as CD spectra using a CD spectropolarimeter (J-500C byJasco) (refer Presta A. et al., Eur. J. Biochem Jan 15;227(1-2):226-240).

The testicular cell differentiation-inducing activity can be detected,for example, as follows. First, spermatogoniums, spermatogenous cells,and primary spermatocytes are isolated from mouse testis bycentrifugation. Next, Tesmin gene is incorporated into an expressionvector (e.g., pBK-CMV vector, Stratagene), and the gene incorporated isintroduced to cells isolated by lipofectAMINE (GIBCO BRL). Afterculturing the cells from a few hours to a few days, the expression of agenetic marker that identifies the differentiation stage (e.g., MEG1,ssH2B, etc.) is verified by the RT-PCR method.

The hybridization technique (Sambrook, J et al., Molecular cloning2^(nd) ed. 9.47-9.58, Cold Spring Harbor Lab. press, 1989) is well knownto a skilled artisan as an alternative method for isolating afunctionally equivalent protein. In other words, it is a generalprocedure for a skilled artisan to isolate DNA having a high homology tothe whole or part of the DNA encoding the mouse or human Tesmin protein(a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to3) and to obtain a protein functionally equivalent to the mouse or humanTesmin protein from the isolated DNA. Therefore, the protein of thepresent invention also includes a protein encoded by DNA hybridizing toDNA encoding the mouse or human-derived Tesmin protein, which isfunctionally equivalent to these proteins. When isolating thehybridizing DNA from other organisms, there is no restriction as to theorganisms used, although testicular tissues from, for example, rats,rabbits, and cattle are suitable for the isolation. DNA isolated byhybridization techniques usually has a high homology to DNA encoding themouse- and human-derived Tesmin protein (DNA comprising the nucleotidesequence of any one of SEQ ID NOs: 1 to 3). “High homology” means, asequence identity at the amino acid level of at least 40% or more,preferably 60% or more, more preferably 80% or more, and even morepreferably, 95% or more. The homology of a sequence can be calculated:for example, by the method described in Proc. Natl. Acad. Sci. USA(1983) 80:726-730.

An example of hybridization conditions (stringent) for isolating a DNAhigh in homology is as follows. Namely, after conducting aprehybridization at 68° C. for 30 min or more using the “Rapid-hybbuffer” (Amersham LIFE SCIENCE), a labeled probe is added, andhybridization is done by incubating at 68° C. for 1 hr or more. Afterthat, washing is done three times within 2×SSC/0.01% SDS for 20 min atroom temperature, and next, three times within 1×SSC/0.1% SDS, at 37° C.for 20 min, followed by, two times within 1×SSC/0.1% SDS, at 50° C. for20 min.

This invention also provides a DNA encoding the Tesmin protein. The DNAof the present invention includes genomic DNA, synthetic DNA, and such,as well as cDNA, as long as such DNA encodes the Tesmin protein of theinvention. The DNA of the invention can be used, for example, forproducing recombinant proteins. Namely, the recombinant proteins can beprepared by inserting the DNA of the invention (e.g., SEQ ID NOs: 1 and2) into a suitable expression vector, introducing this into a suitablecell, culturing the resulting transformant, and purifying the proteinexpressed. Cells used for the production of recombinant proteins are,for example, mammalian cells such as COS cells, CHO cells, and NIH3T3cells; insect cells such as Sf9 cells; yeast cells; and E. coli, butthere is no restriction as to the cells used. The vector for expressingthe recombinant protein within cells varies according to the host cell,and, for example, pcDNA3 (Invitrogen), and pEF-BOS (Nucleic Acids. Res.1990, 18 (17), p5322) and such are given as vectors for mammalian cells,Bac-to-BAC baculovirus expression system (GIBCO BRL) and such for insectcells, Pichia Expression Kit (Invitrogen) and such for yeast cells, andpGEX-5x-1 (Pharmacia) and QIAexpress system (Qiagen) and such for E.coli. Vectors can be introduced into hosts for example, by the calciumphosphate method, DEAE dextran method, the method using cationicliposome DOTAP (Boehringer Mannheim), electroporation method, calciumchloride method, etc. Transformants can be cultured according to theirproperties using methods well known to skilled artisans. Recombinantproteins can be purified from transformants by methods well known toskilled artisans, for example, the methods described in reference “TheQiaexpressionist handbook, Qiagen, Hilden, Germany.”

The present DNA can be used for gene therapy of diseases-caused bymutations of the gene. The Tesmin gene especially may be the causativeof the genetic disease of infertile mice, and therefore, is expected tobe applied in the gene therapy of infertility. When using for genetherapy, the DNA of the invention is inserted into, for example, a viralvector such as an adenovirus vector (e.g. pAdexLcw) and a retrovirusvector (e.g. pZIPneo), or a non-viral vector, and administered to atarget site of the body. The method of administration may be ex vivo orin vivo.

The present invention also provides an antibody that binds to theprotein of the invention. The antibody of the present invention includespolyclonal antibodies and monoclonal antibodies. These antibodies can beprepared by following methods well known to skilled artisans. Polyclonalantibodies can be made by, for example, obtaining the serum of smallanimals such as rabbits immunized with the protein (or a partialpeptide) of the present invention, and purifying by, for example,ammonium sulfate precipitation, a protein A or protein G column, etc.Monoclonal antibodies can be made by immunizing small animals such asmice with the protein (or a partial peptide) of the present invention,excising the spleen from the animal, homogenizing the organ into cells,fusing the cells with mouse myeloma cells using a reagent such aspolyethylene glycol, selecting clones that produce antibodies againstthe protein of the invention from the fused cells (hybridomas),transplanting the obtained hybridomas into the abdominal cavity of amouse, and collecting ascites from the mouse. The obtained monoclonalantibodies can be purified by, for example, ammonium sulfateprecipitation, a protein A or protein G column, etc. The antibody thusprepared can be applied for antibody therapy and such, other than forthe purification and detection of the protein of the invention. Whenadministrating the antibody to humans with the aim of antibody therapy,humanized antibodies are effective in decreasing immunogenicity.Antibodies can be humanized by, for example, cloning the antibody genefrom monoclonal antibody producing cells and using the CDR graft methodwhich transplants the antigen-recognition site of the gene into a knownhuman antibody. Human antibodies can also be prepared like ordinarymonoclonal antibodies by immunizing a mouse whose immune system has beenreplaced by a human immune system with the protein of the invention.

This invention also provides a DNA specifically hybridizing to DNAencoding the Tesmin protein and comprising at least 15 nucleotides. Theterm “specifically hybridizing” as used herein indicates thatcross-hybridization does not significantly occur with DNA encodingproteins other than the Tesmin protein, under the usual hybridizationconditions, preferably under stringent hybridization conditions. SuchDNA can be used as a probe for detecting or isolating DNA encoding theTesmin protein, or as a primer for amplification. Tesmin gene isexpressed only in the testis, and even in the testis, it is expressedfor a limited period. Therefore, the DNA can be used as a testisdifferentiation marker (a test drug). Also, there is a possibility thatthe Tesmin gene is the causative gene of the genetic disease ofinfertile mice, and therefore, the DNA may be used for the testing ofinfertility.

INDUSTRIAL APPLICABILITY

The present invention provides the Tesmin protein comprising ametal-binding site, which is closely associated with the differentiationof testicular cells, and the gene thereof. The Tesmin protein and thegene thereof are involved in the differentiation during spermatogenesis,and Tesmin gene expression is not seen in infertile mice. Therefore,this gene may also be the causative gene of the genetic disease ofinfertile mice. Hence, it is anticipated that gene therapy ofinfertility would be possible by introducing the Tesmin gene into thebody or cells. Moreover, Tesmin is expressed in the testis only, andeven in the testis, the expression is seen only at limited stages.Therefore, Tesmin may also be applied as a test drug for determining thedifferentiation stage of testicular cells. Tesmin is also thought tocontain a metal-binding site similar to metallothionein, and therefore,can also be applied as a metal-poison neutralizing agent similar tometallothionein. It is also expected to be utilized in applied studiessuch as those analyzing the importance of metal binding in the testis.

The following examples are given to illustrate the present invention. Itshould be understood, however, that the present invention is not to belimited to the specific embodiments described in these examples. It willbe apparent to those skilled in the art that various modifications andvariations can be made to the embodiments of the present inventionwithout departing from the spirit or scope of the present invention.Thus, it is intended that the present invention covers othermodifications and variations of this invention within the scope of theappended claims and their equivalents.

EXAMPLE 1 Isolation of Tesmin Gene Fragment Using RT-PCR

The expression of the novel substance WF-1 (a function-unknown novelgene comprising 1700 bp) in each organ was analyzed by the RT-PCRmethod. Specifically, total RNA was extracted from the brain, liver,spleen, kidney, heart, and testis of ICR strain mice (Clea Japan) usingISOGEN (NIPPON GENE). After denaturing RNA at 65° C., cDNA was preparedusing reverse transcriptase: superscript 2 (GIBCO BRL). Using cDNA fromeach organ, and the oligo primers for WF-1 amplification described inSEQ ID NOs: 6 and 7, PCR reaction was conducted for 32 cycles of 94° C.for 1 min, 58° C. for 2 min, and 72° C. for 3 min. The control GAPDH wasamplified by PCR using the oligo primers described in SEQ ID NOs: 8 and9 under the condition of 30 cycles of 94° C. for 1 min, 58° C. for 2min, and 72° C. for 3 min. As a result, a gene specifically expressedonly in the testis was unexpectedly found through a detection usingoligo primers for WF-1 amplification described in SEQ ID NOs: 6 and 7.This cDNA fragment was isolated, and the gene encoded by the cDNA wasnamed “Tesmin” (first named “Testin,” but later changed to “Tesmin”).

EXAMPLE 2 Cloning and Sequencing of Mouse Tesmin cDNA

The sequence of the above cDNA fragment was determined by the dideoxychain termination method and analyzed by the ABI377 auto sequencer. As aresult of a database search for the determined sequence, this sequencewas revealed to be a novel gene that did not have a homology to genes inthe databank. This cDNA fragment was ³²P radio-labeled to prepare aprobe, and using this, a mouse testis library was screened. As a result,a clone having an approximately 1.7 kb length was obtained.

Moreover, 5′-RACE was conducted to determine the 5′ end sequence. In5′-RACE, three antisense primers specific to the Tesmin gene, namely,SP1 (SEQ ID NO: 10), SP2 (SEQ ID NO: 11), and SP3 (SEQ ID NO: 12), andmouse testis-derived 5′-Marathon RACE cDNA were used. The 5′-RACE methodwas conducted following the protocol provided in the Marathon-Ready™cDNA kit (mouse testis), which is commercially available from Clontech.The whole nucleotide sequence of Tesmin cDNA obtained is shown in SEQ IDNOs: 1 (2241 bp) and 2 (1861 bp). These two cDNAs are thought to besplicing variants arising from a difference in splicing at the point oftranscription.

When the database was searched using these cDNA sequences, no sequencecomprising a significant homology was found in the databank. These cDNAsencode the same protein comprising 295 amino acids (pI-7.64), and nosignificantly homologous proteins were found in the protein database aswell.

EXAMPLE 3 Cloning and Sequencing of Human Tesmin cDNA

Mouse Tesmin plasmid (a plasmid in which the Tesmin gene has beeninserted into the pBluescript2 vector) was cleaved by SphI-SalI, andthis 1.7 kb gene fragment was used as a probe to screen the cDNA libraryprepared by human testis mRNA. Hybridization was done using the“Rapid-hyb buffer” (Amersham LIFE SCIENCE) under the followingconditions: (i) a prehybridization at 60° C. for 30 min, (ii) additionof the labeled probe, and (iii) hybridization by incubating at 60° C.for 2 hr. After that, washing is done three times within 2×SSC, 0.01%SDS for 20 min at room temperature, and next, three times within 1×SSC,0.1% SDS, at 37° C. for 20 min, followed by, two times within 1×SSC,0.1% SDS, at 50° C. for 20 min.

The nucleotide sequence of thus obtained human Tesmin cDNA is shown inSEQ ID NO: 3. Database search for the determined nucleotide sequence wasdone but there were no homologous sequences within the databank, similarto the mouse cDNA. The obtained human cDNA had four amino acids morethan mouse Tesmin and encoded a protein comprising 299 amino acids(pI-7.71). No significant homology was found in the protein database aswell. However, as a result of amino acid sequence analysis by BLAST, themouse and human Tesmins were found to be cysteine-rich proteinspartially having the structure very similar to the metal-binding domainof the metallothionein family.

Metallothionein expression in the liver is induced by heavy metals, andmetallothionein is known as a protein that neutralizes metallic poison.However, in the testis, the metallothionein gene is constantly expressedand is not induced by metals. Therefore, it was thought to play somevital roles other than metal binding in the testis. Recent findingsshowed that the estrogen receptor, which is a zinc-finger transcriptionfactor and a receptor protein, and metallothionein conduct metaltransfer in vitro (Cano-Gauci, D. and Sarkar, B. (1996) FEBS Lett 386(1):1-4). Therefore, the metal-binding site of metallothionein isthought to play a vital role in the regulation of transcription factors.The “Cys-X-Cys-X-X-X-X-X-X-X-X-X-Cys-X-Cys (where it is an arbitraryamino acid)” sequence (SEQ ID NO: 23) having a cysteine structure in theamino acid sequence is thought to be vital for metal binding in themetallothionein family.

This cysteine structure (in mouse, from the 157^(th) to the 171^(st)positions, in human, from the 161^(st) to the 175^(th) positions) wasconserved in Tesmin too. However, the metallothionein family membersknown up to now were relatively low-molecular comprising 60 to 70 aminoacids, whereas Tesmin was comparatively longer (mouse: 295 amino acids,human: 299 amino acids). Domain search by PROSITE revealed that mouseTesmin had a N-myristylation site and a casein kinase 2 phosphorylationsite. Human Tesmin had a cAMP and cGMP-dependent kinase phosphorylationsite, a protein kinase C phosphorylation site, a N-myristylation site,and a N-glycosylation site. Other than those, a cAMP and cGMP-dependentprotein kinase phosphorylation site and a protein kinase phosphorylationsite were also present.

Domain search by BLOCKS revealed sites common to mouse and humanTesmins. Namely, “high potential iron-sulfate protein” (from 87^(th) to103^(rd) positions in mouse, from 87^(th) to 103^(rd) positions inhuman), “Adenodoxin family” (iron-sulfate binding region) (from 177^(th)to 194^(th) positions in mouse, from 181^(st) to 198^(th) positions inhuman), “Alpha-2-mavrogloburin family thiolester region” (from 243^(rd)to 252^(nd) positions in mouse, from 247^(th) to 256^(th) in human),“Arrestins proteins” (from 267^(th) to 277^(st) positions in mouse, from271^(st) to 281^(st) positions in human), “Ribosomal protein L14proteins” (from 5^(th) to 26^(th) positions in mouse, from 5^(th) to26^(th) positions in human), “Cooper amine oxidase topaquinone proteins”(from ₈ 1 to 109^(th) positions in mouse, from 81^(st) to 109^(th)positions in human), and “VFWC domain proteins” (from 13^(th) to 19^(th)positions and from 105^(th) to 113^(th) positions in mouse, from105^(th) to 113^(th) positions in human) were confirmed in mouse andhuman Tesmins. When sequence features were analyzed by PRINTS, bothmouse and human Tesmins had a “Rhodopsin-like GPCR superfamilysignature” (from 93^(rd) to 117^(th) positions, from 231^(st) to252^(nd) positions, and from 232^(nd) to 253^(rd) positions in mouse,from 43^(rd) to 67^(th) positions and from 236^(th) to 257^(th)positions in human).

EXAMPLE 4 Transcription and Translation In Vitro

In vitro translation was done to verify the open reading frameanticipated in mouse Tesmin. Specifically, the cDNA pBluescript-Tesmincloned from the testis was transcribed and translated for one hour invitro using the rabbit reticulocyte lysate (Promega Corp.) to whichL-[³⁵S] methionine has been added. Translation products were separatedby SDS-PAGE, and detected by autoradiography. As a result, a protein ofapproximately 32.5 kDa was detected (FIG. 3). This product coincidedwell with the size of the protein thought to be the ORF within the mouseTesmin sequence.

EXAMPLE 5 Preparation of Recombinant Tesmin

The open reading frame of mouse Tesmin cDNA was amplified by a PCRreaction using sense (SEQ ID NO: 19) and antisense (SEQ ID NO: 20)primers having an EcoRI site. The fragment amplified by the PCR reactionwas cloned to the pGEM-T vector to verify its precise sequence. Next, itwas cleaved by EcoRI-EcoRI, and finally cloned to pGEX-2TK vector thatproduces GST fusion protein. Tesmin product cloned into pGEX-2TK wasgene transfected into E. coli JM109, induced using IPTG 0.2 mM at 37° C.for 3 hr, and the E. coli lysate was separated by SDS-PAGE, and detectedby Western blotting using the GST antibody. As a result, a 58.5 kDaprotein comprising a GST fusion portion with a molecular weight of 26kDa was synthesized (FIG. 8 left). The recombinant protein had the samesize expected by the presumed size of the molecule, similar to theresult of in vitro translation.

EXAMPLE 6 Northern Blot Analysis

A membrane loaded with 2 μg/lane of various mouse and human tissue mRNAwas purchased (Clontech laboratories, Palo Alto, Calif.), and Northernblot analysis was conducted. The probe was a 1.7 kb gene fragment madeby cleaving mouse Tesmin plasmid (a plasmid in which the Tesmin gene hasbeen inserted into pBluescript2 vector) with SphI-Sal I. Hybridizationwas done using the “Rapid-hyb buffer” (Amersham LIFE SCIENCE) under thefollowing conditions: (i) a prehybridization at 68° C. for 30 min, (ii)addition of the labeled probe, and, (iii) hybridization by incubating at68° C. for 2 hr. Next, washing is done three times within 2×SSC, 0.01%SDS for 20 min at room temperature, and next, three times within 1×SSC,0.1% SDS, at 37° C. for 20 min, followed by, two times within 1×SSC,0.1% SDS, at 50° C. for 20 min. Detection was done by autoradiography.Similar to the results of RT-PCR, Tesmin gene expression was detectablein the testis only, and gene expression was seen at the 2.4 kb and 2.0kb locations in mouse (FIG. 1) and in just the 2.4 kb location in human(FIG. 2).

EXAMPLE 7 Involvement in the Differentiation of Reproductive Cells

Total RNA was extracted from the testis of ICR strain mouse at day 4, 8,12, 18, and 42 following birth, and from the day 56 testis of W/Wvstrain mouse (Japan SLC; type WBB6F1-W/Wv known as an infertile mousedeficient of the mouse growth factor S1 receptor c-kit gene; referChabot, B. et al. (1988) Nature 335 (6185):88-9, Yoshinaga, K. et al.(1991) Development 113 (2):689-99) using ISOGEN (NIPPON GENE). Afterdenaturing this RNA at 65° C., cDNA was prepared using reversetranscriptase: superscript 2 (GIBCO BRL). Tesmin gene was amplifiedusing the oligo primers described in SEQ ID NOs: 6 and 7, under theconditions of 35 cycles of 94° C. for 1 min, 58° C. for 2 min, and 72°C. for 3 min. The control GAPDH gene was amplified using the oligoprimers described in SEQ ID NOs: 8 and 9, under the conditions of 30cycles of 94° C. for 1 min, 58° C. for 2 min, and 72° C. for 3 min. Forthe MEG1 PCR reaction, the oligo primers described in SEQ ID NOs: 13 and14 were used, under conditions of 35 cycles of 94° C. for 1 min, 58° C.for 2 min, and 12° C. for 3 min. For the ssH2B reaction, the oligoprimers described in SEQ ID NOs: 15 and 16 were used, under theconditions of 35 cycles of 94° C. for 1 min, 58° C. for 2 min, and 72°C. for 3 min. Marker MEG1 expresses when spermatogenous cells divideinto primary spermatocytes (Don, J. and Wolgemuth, D. J. (1992) August;3 (8):495-505), and marker ssH2B is known to express at spermatogenesis(Unni, E. et al. (1995) Biol Reprod, October; 53 (4):820-826).

PCR analysis showed that Tesmin gene is not expressed until day 8following birth, having a weak expression at day 12, and taking a stableexpression pattern from day 18 (FIG. 4). Tesmin gene expression patternin the testis was similar to MEG1. Therefore, it was revealed thatTesmin expression is regulated at time points similar to MEG1.

When Tesmin expression in the W/Wv mouse was examined, it was revealedthat Tesmin is not expressed in these mice. Since the W/Wv mouse isknown to be an infertile mouse, the relationship between Tesmin gene andinfertility was strongly suggested (FIG. 4).

EXAMPLE 8 In Situ Hybridization

Labeled RNA probe was prepared from mouse Tesmin plasmid (thepBluescript2 vector in which the Tesmin gene has been inserted) using T7and T3 polymerases and digoxigenin-duTP. This probe was hybridized tosliced mouse testis tissue within a solution containing 50% formamide,10% dextran sulfate, and 2×SSC. The slide glass on which hybridizationwas done was incubated within a solution of anti-digoxigenin antibodybound to alkaline phosphatase, and the signal specific to hybridizationwas detected using the chromogenic substrate NBT/BCIP. As a result, itwas verified that Tesmin is extremely specifically expressed in thetestis, especially in primary spermatocytes (FIG. 5).

EXAMPLE 9 Chromosomal Location

Mouse P1 genomic library was obtained by PCR screening the P1bacteriophage genomic library using a mouse Tesmin-specific sense primer(SEQ ID NO: 6) and an antisense primer (SEQ ID NO: 7).Also, human P1genomic library was obtained using human Tesmin-specific sense primer(SEQ ID NO: 17) and antisense primer (SEQ ID NO: 18) and conducting ascreening similar to mouse. The isolated P1 clones were used to examinethe chromosomal localization by fluorescent in situ hybridization(FISH). Mouse and human P1 clone-derived DNA was labeled by nicktranslation using digoxigenin-dUTP, and this probe was hybridized tomouse and human primary fibroblast-derived metaphase chromosomes withina solution containing 50% formamide, 10% dextran sulfate, and 2×SSC. Theslide glass on which hybridization was done was incubated within asolution of fluorescence-labeled anti-digoxigenin antibody, and thesignal specific to hybridization was detected by counter staining using4′6′-diamino-2-phenolindol (DAPI).

As a result, the above P1 clones were found to encode the Tesmin genesince the mouse and human Tesmin-specific probes hybridized to therespective P1 clone. When DAPI staining was done using these P1 clonesas probes, the 19^(th) B chromosome and the 11^(th) q 13.2 chromosomewere specifically labeled in mouse and human, respectively. The aboveresults confirmed that Tesmin was located on the 19^(th) B chromosome(FIG. 6) in mouse, and on the 11^(th) q13.2 chromosome (FIG. 7) inhuman. The relationship between Tesmin and mouse genetic disease wasexamined based on these results using the Jackson Laboratory Database tofind that there is a study reporting that a mutation on the 19^(th) Bchromosome where Tesmin exists causes infertility in mice (Evans, E P.(1977) Mouse News Letter, 17). This suggests the possibility that Tesminmutations trigger infertility in mice.

EXAMPLE 10 Intracellular Localization

A DNA encoding whole open reading frame of the Tesmin cDNA wereprepared, using sense (SEQ ID NO: 19) and antisense (SEQ ID NO: 20)primers having an EcoRI site, and also prepared was a gene designed sothat 70 amino acids are deleted from the Tesmin cDNA open reading frame,by using a sense (SEQ ID NO: 19) primer having an EcoRI site andantisense (SEQ ID NO: 21) primer having an SalI site. These genes weretreated with restriction enzymes and inserted into the C terminal regionof GFP ORF of the pEGFC1 vector (Clontech). Using Tfx-50 (Promega), thisplasmid that encodes the GFP-Tesmin fusion protein was introduced intoCOS1 cells growing on a cover glass. The cover glass was fixed bymethanol/acetone (1:1) and washed three times with PBS. The cells wereobserved with Olympus BH-2 Epifluorescent Microscope. As a result,although the protein fused to the full sequence of Tesmin was localizedwithin the cytoplasm, one having the partially deleted Tesmin sequencehad migrated into the nucleus (FIG. 8).

EXAMPLE 11 Preparation of a Specific Antibody that Binds to the TesminProtein

A peptide antibody against the 18 amino acids presumed by the genearrangement of Tesmin was prepared. Specifically, an 18 amino acidsequence (SEQ ID NO: 22) was made using a peptide synthesizer. KLH wascovalently bound to this obtained peptide with a crosslinking reagent.Next, this peptide was purified by HPLC, and a rabbit was immunized withit. Serum was drawn out at four stages, and finally, all the blood wascollected. This serum was purified, using a protein A column to preparethe polyclonal antibody. Tesmin protein fused with GST was separated ona gel by SDS-PAGE. Detection by Western blotting confirmed that thisanti Tesmin polyclonal antibody recognizes the Tesmin protein (FIG. 9).

Western blotting was done by inducing recombinant protein expressionthrough isopropyl-β-D-thiogalactoside (IPTG) added toTesmin-cDNA-introduced E. coli, and subjecting an E. coli lysate toSDS-PAGE (IPTG+, FIG. 9). A detection using a cell lysate of E. coliwithout IPTG was also done (IPTG−, FIG. 9).

1. A method of producing a protein comprising: culturing a transformantcomprising a DNA encoding a protein comprising an amino acid sequence inwhich one of several amino acids of SEQ ID NOs: 4 or 5 have beenreplaced, deleted, and/or added, and which is functionally equivalent toa protein comprising SEQ ID NOs: 4 or 5 under conditions wherein aprotein is expressed, wherein the protein comprises an amino acidsequence in which one of several amino acids of SEQ ID NOs: 4 or 5 havebeen replaced, deleted, and/or added, and wherein the protein isfunctionally equivalent to a protein comprising SEQ ID NOs: 4 or 5; andobtaining the protein from the transformant or the culture supernatantthereof.
 2. A vector comprising a DNA encoding a protein comprising anamino acid sequence in which one of several amino acids of SEQ ID NOs: 4or 5 have been replaced, deleted, and/or added, and which isfunctionally equivalent to a protein comprising SEQ ID NOs: 4 or
 5. 3. Avector comprising a DNA encoding a protein which is encoded by a DNAhybridizing under stringent conditions to the DNA comprising thenucleotide sequence of SEQ ID NOs: 1 or 3, wherein the protein isfunctionally equivalent to a protein comprising SEQ ID Nos: 4 or 5.